Abstract

A strategy is presented to enable optical-sectioning microscopy with improved contrast and imaging depth using low-power (0.5 - 1 mW) diode laser illumination. This technology combines the inherent strengths of focal-modulation microscopy and dual-axis confocal (DAC) microscopy for rejecting out-of-focus and multiply scattered background light in tissues. The DAC architecture is unique in that it utilizes an intersecting pair of illumination and collection beams to improve the spatial-filtering and optical-sectioning performance of confocal microscopy while focal modulation selectively ‘labels’ in-focus signals via amplitude modulation. Simulations indicate that modulating the spatial alignment of dual-axis beams at a frequency f generates signals from the focal volume of the microscope that are modulated at 2f with minimal modulation of background signals, thus providing nearly an order-of-magnitude improvement in optical-sectioning contrast compared to DAC microscopy alone. Experiments show that 2f lock-in detection enhances contrast and imaging depth within scattering phantoms and fresh tissues.

Principles of modulated-alignment dual-axis (MAD) confocal microscopy. By periodically sweeping the illumination beam into (a) and out of alignment (b) with the collection beam at a given frequency f, the signal generated at the focal volume experiences an amplitude modulation at 2f (c) and can therefore be distinguished from background signal that remains largely unmodulated.

MAD architecture and optical circuit. (a) The MAD system is built by modifying a typical dual-axis confocal (DAC) tabletop microscope. An acousto-optical deflector (AOD), which is optimized to produce a 1st-order diffracted beam, is inserted into the optical path upstream of the illumination arm. The 1st diffracted order serves as the illumination beam in the MAD system. A slit (S1) oriented perpendicularly to the dispersion direction is used to isolate the 1st-order beam and to block all other diffracted orders. Pixel intensities correspond to the magnitude of the 2f signal. (b) A photograph of the tabletop MAD setup. (c) Motion of the illumination beam is minimized in relation to the motion of the focus (right side of ray-trace diagram) by ensuring that the pivot point about which the illumination beam rotates (for alignment modulation) is located near or at the focusing objective. This ensures that the motion of the illumination beam at the focus is larger than the motion of the beam prior to the focus.

Optimizing modulation depth. (a) In response to a mirror placed at the focal plane, the optical throughput of the microscope system decays as the alignment offset between the illumination and detection paths increases. (b) When sinusoidally modulating the alignment offset at a modulation depth of amplitude Δy/ω0, the maximum 2f signal is generated at Δy/ω0 ~1.5 - 1.8. Example time traces (c) and their frequency content (d) at three distinct modulation depths demonstrate the dependency of the 2f signal on modulation depth.

Monte-Carlo simulations. (a) Typical DAC microscope system geometry where θ is the crossing half-angle between the beams and α is the focusing half-angle of each beam. (b) A ~9-dB improvement in optical-sectioning contrast is achieved at all depths (perpendicular optical lengths. Lp, ranging from 2 to 10). (c) Monte-Carlo simulations show that the optimal modulation depth for maximizing contrast (signal to background ratio, SBR) is consistently Δy/ω0 ~1.5 - 1.8 over a range of imaging depths (or perpendicular optical lengths, Lp).

Axial and transverse responses of MAD and DAC in scattering media. Intralipid experiments demonstrate consistent improvement in optical sectioning contrast in the MAD technique over DAC. (a) Contrast improves by 5 - 6 dB axially without compromising optical section thickness. (b) In-plane resolution is maintained while improving contrast by up to ~4 dB. The signal labeled as 'Water' refers to characterization of the system in the absence of scattering (whereas scattering is achieved using Intralipid).